【摘要】：目的:以殼聚糖為基質(zhì)材料,制備淋球菌porB基因緩釋微球,通過鼻黏膜途徑及肌注途徑免疫BALB/c小鼠,分析其免疫活性,探討其能否增強(qiáng)特異性體液免疫和細(xì)胞免疫應(yīng)答水平,為殼聚糖納米粒作為淋球菌DNA疫苗載體的應(yīng)用提供實(shí)驗(yàn)依據(jù)。 方法:采用復(fù)凝聚法制備CS-pcDNA3.1(-)/porB微球,分別用透射電鏡,激光粒度分析儀觀察微球形態(tài)、大小、zeta電位;核酸酶研究其保護(hù)性; DNA電泳阻滯分析CS-pcDNA3.1(-)/porB納米粒結(jié)合能力;釋放實(shí)驗(yàn)評價CS-pcDNA3.1(-) /porB的穩(wěn)定性;用MTT法評價CS-pcDNA3.1(-)/porB的細(xì)胞毒性高低。將殼聚糖-pcDNA3.1(-)/porB納米粒和裸質(zhì)粒pcDNA3.1(-)/porB納米粒通過鼻飼和肌注途徑免疫雌性BALB/c小鼠,ELISA法測定小鼠體液免疫和細(xì)胞免疫應(yīng)答水平。 結(jié)果: (1)殼聚糖與pcDNA3.1(-)/porB質(zhì)粒形成穩(wěn)定的微球,包封率大于90%,多數(shù)成球形,直徑在100-300nm之間。 (2)殼聚糖納米粒能有效抵抗核酸酶的降解作用,4℃保存45d,殼聚糖納米粒仍能較穩(wěn)定地和porB質(zhì)粒結(jié)合。MTT毒性試驗(yàn)證明殼聚糖-pcDNA3.1(-)/porB納米粒在高劑量才出現(xiàn)低細(xì)胞毒性。 (3) CS-pcDNA3.1(-)/porB與裸質(zhì)粒pcDNA3.1(-)/porB組通過鼻黏膜免疫及肌肉注射免疫均能誘導(dǎo)小鼠產(chǎn)生有效的免疫應(yīng)答。CS-pcDNA3.1(-)/porB誘導(dǎo)的血清特異性IgG和生殖道灌洗液特異性sIgA抗體水平隨免疫時間呈上升趨勢,與裸質(zhì)粒pcDNA3.1(-)/porB組相比差異顯著(P0.05),同時CS-pcDNA3.1(-)/porB鼻飼和肌注免疫組小鼠脾淋巴細(xì)胞經(jīng)特異性抗原刺激后,脾淋巴細(xì)胞誘生的IFN-γ和IL-4含量明顯升高,與裸質(zhì)粒pcDNA3.1(-)/porB組、PBS和殼聚糖對照組之間差異具有顯著性(P0.01);脾淋巴細(xì)胞刺激指數(shù)(分別為1.54±0.28和1.63±0.27)明顯高于裸質(zhì)粒pcDNA3.1(-)/porB組、殼聚糖組和PBS組(P 0.05 );鼻飼途徑的CS-pcDNA3.1(-)/porB組、裸質(zhì)粒pcDNA3.1(-)/porB組血清IgG亞類IgG2a/IgG1比值分別為1.441、1.745 ,肌注途徑的CS-pcDNA3.1(-)/porB組、裸質(zhì)粒pcDNA3.1(-)/porB組血清IgG亞類IgG2a/IgG1比值分別為1.387、1.761。 結(jié)論: (1) porB基因可與殼聚糖形成穩(wěn)定的緩釋微球,并能在293T細(xì)胞內(nèi)表達(dá); (2)殼聚糖納米粒能增強(qiáng)pcDNA3.1(-)/porB核酸疫苗的鼻飼及肌注免疫效果; (3) pcDNA3.1(-)/porB核酸疫苗主要誘導(dǎo)Th1型傾向性免疫應(yīng)答。
[Abstract]:Objective: to prepare sustained release microspheres of porB gene from Neisseria gonorrhoeae and immunize BALB/c mice by nasal mucosal pathway and intramuscular injection. To explore whether it can enhance specific humoral immunity and cellular immune response, and to provide experimental basis for the application of chitosan nanoparticles as DNA vaccine carrier of Neisseria gonorrhoeae (Neisseria gonorrhoeae). Methods: CS-pcDNA3.1 (-) / porB microspheres were prepared by complex coacervation method. The morphology, size and zeta potential of CS-pcDNA3.1 (-) / porB microspheres were observed by transmission electron microscope (TEM) and laser particle size analyzer respectively. The binding ability of CS-pcDNA3.1 (-) / porB nanoparticles was analyzed by DNA electrophoresis, the stability of CS-pcDNA3.1 (-) / porB was evaluated by release assay, and the cytotoxicity of CS-pcDNA3.1 (-) / porB was evaluated by MTT assay. Chitosan-pcDNA3.1 (-) / porB nanoparticles and bare plasmid pcDNA3.1 (-) / porB nanoparticles were used to immunize female BALB/c mice by nasal feeding and intramuscular injection. Humoral and cellular immune responses were measured by ELISA method. Results: (1) chitosan and pcDNA3.1 (-) / porB plasmids formed stable microspheres, the encapsulation efficiency was greater than 90, most of them were spherical, and the diameters were between 100-300nm. (2) chitosan nanoparticles could effectively resist the degradation of nuclease and were stored at 4 鈩，

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